Cyclized fluorescent nucleic acid intercalating cyanine dyes and nucleic acid detection methods

ABSTRACT

New intercalating cyanine dyes are provided in which the benzothiazole portion of the cyanine dye has been modified to produce dyes with improved properties for labelling nucleic acids. These new intercalating cyanine dyes are cyclized fluorescent cyanine dyes represented by General Formula I ##STR1## where: n is 0, 1 or 2; 
     Y may be either S or O; 
     R 3  and R 4  may each independently be either hydrogen, C 1  -C 10  alkyl, C 1  -C 10  alkoxy or C 1  -C 10  alkylthio; 
     R 5  may be C 1  -C 50  alkyl, preferably substituted with one or more polar substituents which preferably includes one or more positively charged atoms, or a cyclized fluorescent cyanine dye of the present invention, i.e., where R 5  is a linker between two cyclized fluorescent cyanine dyes; 
     R 6  and R 7  may each independently be either H and C 1-10  alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C 1-6  alkyl or C 1  -C 10  alkoxy groups; 
     R 8  and R 9  may each independently be either H and C 1-10  alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C 1-6  alkyl or C 1  -C 10  alkoxy groups; and 
     R 10  may be either H, C 1-6  alkyl, C 1  -C 10  alkoxy or a fused benzene. 
     As used above, alkyl and alkoxy refer to any substituent having a carbon backbone having the specified range of carbon atoms. The carbon backbone may form a straight chain, may be branched or may be cyclic. The alkyl and alkoxy groups may be optionally substituted by a wide variety of substituents including, for example, alcohols, amines, thiols, phosphates, halides, ethers, esters, ketones, aldehydes, carboxylic acids, amides, cycloalkyls, and aromatic rings.

FIELD OF THE INVENTION

The present invention relates generally to dyes for labelling nucleicacids. More specifically, the present invention relates to intercalatingcyanine dyes for the detection and enumeration of nucleic acids.

BACKGROUND Of THE INVENTION

Intercalating dyes which exhibit enhanced fluorescence upon binding toDNA or RNA are a basic tool in molecular and cell biology. In general,intercalating dyes bind noncovalently to DNA through a combination ofhydrophobic interactions with the DNA base-pairs and ionic binding tothe negatively charged phosphate backbone. The fluorescence of the dyeis ideally increased several-fold upon binding to DNA, thereby enablingthe detection of small amounts of nucleic acids. Examples of fluorescentnoncovalent DNA binding dyes include ethidium bromide which is commonlyused to stain DNA in agarose gels after gel electrophoresis, andpropidium iodide and Hoechst 33258 which are used in flow cytometry todetermine the DNA ploidy of cells.

Fluorescent nucleic acid labelling dyes preferably absorb light betweenabout 300 and 900 nm and preferably have a Stokes shift of at leastabout 10 nm. Dyes that absorb light in the 500 to 900 nm range arepreferred because they are spectrally removed from other components thatmay be present in a biological sample and because they may be used withinexpensive light sources. Fluorescent dyes that have a high extinctioncoefficient, a high quantum yield, and significantly enhancedfluorescence when bound to a nucleic acid are also preferred.

Few new dye chromophores were described until the introduction ofThiazole Orange as a reticulocyte stain in 1986. Lee, et al., "ThiazoleOrange: A New Dye for Reticulocyte Analysis", Cytometry 1986 7, 508-517.Thiazole Orange is an asymmetric cyanine dye. Although many asymmetriccyanine dyes have been described in the art (e.g., Lincoln, et al., U.S.Pat. No. 3,282,932), Thiazole Orange's fluorescence properties whenbound to DNA and RNA and its utility for labelling nucleic acids had notbeen previously identified. Lee, et al., U.S. Pat. No. 4,957,870. Forexample, unlike most asymmetric cyanine dyes, Thiazole Orange exhibits aseveral thousand-fold enhancement in fluorescence upon binding to DNA.

Since the discovery of Thiazole Orange as a nucleic acid dye, severalimprovements to Thiazole Orange and its trimethine homologs have beendeveloped to provide dyes with tighter binding to DNA and greater watersolubility. Xue, et al. U.S. Pat. No. 5,321,130 and Glazer, et al. U.S.Pat. No. 5,312,921. These dyes generally involve a modification to thequinolinium portion of the dye.

A continuing need exists for new and improved dyes for labelling nucleicacids. In particular, a need exists for dyes which exhibit longerwavelengths and significantly enhanced fluorescence when bound to DNA orRNA.

SUMMARY OF THE INVENTION

The present invention relates asymmetric cyanine dyes for non-covalentlylabelling nucleic acids in which the benzothiazole portion of the dyehas been modified to provide improved physical properties to the dye,such as longer wavelengths and improved fluorescence enhancement whenbound to DNA or RNA.

More specifically, the invention relates to cyclized fluorescent cyaninedyes for non-covalently labelling nucleic acids. The cyclizedfluorescent cyanine dyes according to the present invention arerepresented by General Formula I ##STR2## where: n is 0, 1 or 2;

Y may be either S or O;

R₁ and R₂ are taken together to form a 5, 6, 7 or 8 membered ring;

R₃ and R₄ may each independently be either hydrogen, C₁ -C₁₀ alkyl, C₁-C₁₀ alkoxy, or C₁ -C₁₀ alkylthio;

R₅ may be a C₁ -C₅₀ alkyl, preferably substituted with one or more polarsubstituents which preferably includes one or more positively chargedatoms, or a cyclized fluorescent cyanine dye of the present invention,i.e., where R₅ is a linker between two cyclized fluorescent cyaninedyes;

R₆ and R₇ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups;

R₈ and R₉ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups; and

R₁₀ may be either H, C₁₋₆ alkyl, C₁ -C₁₀ alkoxy or a fused benzene.

As used above, alkyl and alkoxy refer to any substituent having a carbonbackbone having the specified range of carbon atoms. The carbon backbonemay form a straight chain, may be branched or may be cyclic. The alkyland alkoxy groups may be optionally substituted by a wide variety ofsubstituents including, for example, alcohols, amines, thiols,phosphates, halides, ethers, esters, ketones, aldehydes, carboxylicacids, amides, cycloalkyls, and aromatic rings.

The invention also relates to the composition of a cyanine dye accordingto the present invention non-covalently bound to a nucleic acidsequence, i.e., RNA or DNA, which enables the nucleic acid sequence tobe analytically detected.

The invention also relates to a method for detecting nucleic acids in asample by contacting the nucleic acids with a fluorescent cyanine dyeaccording to the present invention and monitoring the change influorescence emission of the dye.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates asymmetric cyanine dyes for non-covalentylabelling nucleic acids in which the benzothiazole portion of the dyehas been modified to provide improved physical properties to the dye,such as longer wavelengths and improved fluorescence enhancement whenbound to DNA or RNA.

In one embodiment, the present invention relates to cyclized fluorescentcyanine dyes generally represented by General Formula I ##STR3## where:n is 0, 1 or 2;

Y may be either S or O;

R₁ and R₂ are taken together to form a 5, 6, 7 or 8 membered ring;

R₃ and R₄ may each independently be either hydrogen, C₁ -C₁₀ alkyl, C₁-C₁₀ alkoxy, or C₁ -C₁₀ alkylthio;

R₅ may be a C₁ -C₅₀ alkyl, preferably substituted with one or more polarsubstituents which preferably includes one or more positively chargedatoms, or a cyclized fluorescent cyanine dye of the present invention,i.e., where R₅ is a linker between two cyclized fluorescent cyaninedyes;

R₆ and R₇ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups;

R₈ and R₉ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups; and

R₁₀ may be either H, C₁₋₆ alkyl, C₁ -C₁₀ alkoxy or a fused benzene.

As used above, alkyl and alkoxy refer to any substituent having a carbonbackbone having the specified range of carbon atoms. The carbon backbonemay form a straight chain, may be branched or may be cyclic. The alkyland alkoxy groups may be optionally substituted by a wide variety ofsubstituents including, for example, alcohols, amines, thiols,phosphates, halides, ethers, esters, ketones, aldehydes, carboxylicacids, amides, cycloalkyls, and aromatic rings.

The cyclized cyanine dyes of the present invention provide the advantageover previous cyanine dyes of having higher absorbance and emissionwavelengths. The cyclized cyanine dyes preferably absorb light at awavelength of at least about 640 nm, more preferably at least about 649nm and emit fluorescence at a wavelength of at least about 650 nm, morepreferably at least about 663 nm. The cyclized cyanine dyes alsopreferably have a positive Stoke's shift (λ_(Emission) -λ_(Abs).) of atleast about 12 nm.

In particular, cyclized cyanine dyes having General Formula I where R₁and R₂ are taken together to form a 5, 6, 7 or 8 membered ring have beenfound to absorb light and fluoresce when bound to a nucleic acid polymerat unexpectedly higher wavelengths than has been previously achieved bycyanine dyes where R₁ and R₂ do not form a ring structure.

Fluorescent cyanine dyes having the General Formula I where R₁ and R₂are taken together to form a 7 membered ring have also been found tohave the greatest Stoke's shift (λ_(Emission) -λAbs.).

                                      TABLE 1    __________________________________________________________________________    Absorbance and Emission Maxima of Intercalating Dyes    in PBS with Excess DNA ([bp]/[dye] = 100)    COMPOUND                             Abs.sub.max                                              Ems.sub.max                                                   F.E.    __________________________________________________________________________     ##STR4##                        1   649  663  100X     ##STR5##                        2   654  667  100X     ##STR6##                        3   654  672   30X     ##STR7##                        4   675  690  200X     ##STR8##                        5*  641  655  100X    __________________________________________________________________________     Abs.sub.max -- Absorbance maximum (bound to DNA)     Ems.sub.max -- Emission maximum (bound to DNA)     F.E. -- fluorescence enhancement (bound vs. not bound to DNA or RNA)     *Compound 5 is taught in U.S. Pat. No. 5,321,130 to Yue, et al.

Table 1 summarizes the absorbance maximum and fluorescence emissionmaximum wavelengths (both when bound to DNA) of some exemplary cyclizedcyanine dyes of the present invention.

As illustrated in Table 1, it was found that the addition of a cyclicaliphatic side chain to the basic cyanine dye structure, i.e., formationof a 5-8 membered ring by combining R₁ and R₂, was found to increase theabsorbance and fluorescence emission wavelengths of the correspondingacyclic cyanine dye by about 12 nm. For example, as shown with regard todyes 2 and 5, dye 2 has an Abs_(max) at 654 nm as compared to 641 nm andan Ems_(max) at 667 nm as compared to 655 nm. In addition, dye 4 is thelongest wavelength trimethine intercalating dye yet reported.

With regard to n, n may equal 1. Accordingly, the present inventionincludes cyclized cyanine dyes having the General Formula II (i.e. wheren=1) ##STR9## where Y, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are asspecified above.

Y may be either S or O, and is most preferably S.

R₃ and R₄ may each independently be either hydrogen, C₁ -C₁₀ alkyl, C₁-C₁₀ alkoxy, or C₁ -C₁₀ alkylthio, and are preferably H.

R₅ may be a C₁ -C₅₀ alkyl. Since DNA and RNA to which the cyclizedcyanine dyes bind contain negatively charged phosphate backbones, it ispreferred that R₅ be substituted with one or more polar substituents. Itis most preferred that R₅ include one or more positively charged atomsin the polar substituent. U.S. Pat. No. 5,321,130 to Yue, et al. teachesunsymmetrical cyanine dyes having an aminoalkyl chain containing abackbone of 3-42 carbons and 1-5 positively charged nitrogen atoms. Thecationic tail described in U.S. Pat. No. 5,321,130 exemplifies one ofthe positively charged R₅ substituents that may be used in combinationwith the cyclic cyanine dyes of the present invention and isincorporated herein by reference. In addition to the positively chargedR₅ substituents described in U.S. Pat. No. 5,321,130, R₁₂ is alsointended to include aminoalkyl chains including a positively chargedcyclic aminoalkyl group having 1-5 positively charged nitrogen atoms.

Alternatively, R₅ may form part of a linker between two cyclizedfluorescent cyanine dyes as illustrated by General Formula IV ##STR10##

According to this embodiment, Y, R₁, R₂, R₃, R₄, R₆, R₇, R₈, R₉ and R₁₀are as specified above. It should be noted that the two linked cyaninedyes may be the same or different cyanine dyes. In general, it ispreferred that the linked cyanine dyes be the same since different dyeswill have different spectral properties.

R₆ and R₇ may each independently be either H, C₁₋₁₀ alkyl, or are takentogether to form a 5 or 6 membered ring, most preferably a 5 or 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups.

R₈ and R₉ may each independently be either H, C₁₋₁₀ alkyl, or are takentogether to form a 5 or 6 membered ring, most preferably a 5 or 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups.

In general, it is preferred either R₆ and R₇ or R₈ and R₉ are takentogether to form a 5 or 6 membered aromatic ring, optionally substitutedwith C₁₋₆ alkyl or C₁ -C₁₀ alkoxy groups. The R₆ and R₇ or R₈ and R₉groups that do not form the aromatic ring are preferably H.

R₁₀ may be either H, C₁₋₆ alkyl, C₁ -C₁₀ alkoxy or a fused benzene.

In a particularly preferred embodiment, the cyclized cyanine dye has theGeneral Formula V where the ring formed by R₁ and R₂ includes apositively charged substituent R₂₇. As discussed herein, inclusion of apositively charged substituent, such as R₂₇, to a substituent on thepositively charged nitrogen on the benzothiazole ring improves the netfluorescence enhancement of the dye with DNA. ##STR11##

R₂₇ is a positively charged alkyl substituent which may be attached toany atom used to form the 5, 6, 7 or 8 membered ring. R₂₇ is morepreferably a positively charged aminoalkyl substituent. For example, R₁₂can be an aminoalkyl chain containing a backbone of 3-42 carbons and 1-5positively charged nitrogen atoms as described in U.S. Pat. No.5,321,130 to Yue, et al. which is incorporated herein by reference. Inaddition to the positively charged substituents described in U.S. Pat.No. 5,321,130, R₁₂ is also intended to include aminoalkyl chainsincluding a positively charged cyclic aminoalkyl group having 1-5positively charged nitrogen atoms.

In a preferred embodiment, R₂₇ has the general formula --R₂₈ N(R₂₉ R₃₀R₃₁) where R₂₈ is a C₁₋₅ alkyl and R₂₉, R₃₀, and R₃₁ are eachindependently a C₁₋₁₀ alkyl.

Table 2 provides examples of some of the preferred cyclized cyaninedyes. It should be understood, however, that the dyes listed in Table 2are intended only to exemplify the cyclized cyanine dyes of the presentinvention and are not intended to be limiting.

    TABLE 2      -      ##STR12##      ##STR13##      ##STR14##      ##STR15##      ##STR16##      ##STR17##      ##STR18##      ##STR19##      ##STR20##      ##STR21##      ##STR22##      ##STR23##      ##STR24##      ##STR25##      ##STR26##      ##STR27##

The present invention also relates to fluorescent cyanine dyes having apositively charged substituent attached to the positively chargednitrogen on the benzothiazole portion of the cyanine dye. Thesefluorescent cyanine dyes are represented by General Formula VI ##STR28##where n is 0, 1 or 2;

Y may be either S or O;

R₁₂ is a positively charged alkyl substituent, more preferably apositively charged aminoalkyl substituent;

R₁₃, R₁₄ and R₁₅ may each independently be either hydrogen, C₁ -C₁₀alkyl, C₁ -C₁₀ alkoxy, or C₁ -C₁₀ alkylthio;

R₁₂ and R₁₃ may optionally be taken together to form a 5, 6, 7 or 8membered ring;

R₁₆ may be a C₁ -C₅₀ alkyl, preferably substituted with one or morepolar substituents which preferably includes one or more positivelycharged atoms, or a cyclized fluorescent cyanine dye of the presentinvention, i.e., where R₁₆ is a linker between two cyclized fluorescentcyanine dyes;

R₁₇ and R₁₈ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 5 or 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups;

R₁₉ and R₂₀ may each independently be either H or C₁₋₁₀ alkyl, or may betaken together to form a 5 or 6 membered ring, most preferably a 5 or 6membered aromatic ring, optionally substituted with C₁₋₆ alkyl or C₁-C₁₀ alkoxy groups; and

R₂₁ may be either H, C₁₋₆ alkyl, C₁ -C₁₀ alkoxy or a fused benzene.

As used above, alkyl and alkoxy refer to any substituent having a carbonbackbone having the specified range of carbon atoms. The carbon backbonemay form a straight chain, may be branched or may be cyclic. The alkyland alkoxy groups may be optionally substituted by a wide variety ofsubstituents including, for example, alcohols, amines, thiols,phosphates, halides, ethers, esters, ketones, aldehydes, carboxylicacids, amides, cycloalkyls, and aromatic rings.

With regard to n, it is noted that n may equal 1. Accordingly, anembodiment of the present invention includes cyanine dyes having theGeneral Formula VII (i.e. where n=1) ##STR29## where Y, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀ and R₂₁ are as specified above.

With regard to dyes having General Formula VI or VII, Y may be either Sor O and is most preferably S.

R₁₂ can be an aminoalkyl chain containing a backbone of 3-42 carbons and1-5 positively charged nitrogen atoms as described in U.S. Pat. No. 55,321,130 to Yue, et al. which is incorporated herein by reference. Inaddition to the positively charged substituents described in U.S. Pat.No. 5,321,130, R₁₂ is also intended to include aminoalkyl chainsincluding a positively charged cyclic aminoalkyl group having 1-5positively charged nitrogen atoms.

In a preferred embodiment, R₁₂ has the general formula --R₂₈ N(R₂₉ R₃₀R₃₁) where R₂₈ is a C₁₋₅ alkyl and R₂₉, R₃₀, and R₃₁ are eachindependently a C₁₋₁₀ alkyl.

In an alternate preferred embodiment, R₁₂ and R₁₃ are taken together toform a 5, 6, 7 or 8 membered ring where the ring includes a positivelycharged alkyl substituent, more preferably an aminoalkyl chaincontaining a backbone of 3-42 carbons and 1-5 positively chargednitrogen atoms as described in U.S. Pat. No. 5,321,130 to Yue, et al.Dyes of this embodiment may be generally represented by General FormulaVIII ##STR30## where R₁₂ and R₁₃ represents the atoms necessary to forma 5, 6, 7 or 8 membered ring and R₂₇ is a positively chargedsubstituent, as specified above with regard to R₁₂, which may beattached to any atom used to form the 5, 6, 7 or 8 membered ring asrepresented by R₁₂ and R₁₃. In this regard, these dyes are equivalent tothe dyes described above having the General Formula V.

R₁₄ and R₁₅ may each independently be either hydrogen, C₁ -C₁₀ alkyl, C₁-C₁₀ alkoxy, or C₁ -C₁₀ alkylthio, and are preferably H.

R₁₆ may be a C₁ -C₅₀ alkyl. Since DNA and RNA to which the cyclizedcyanine dyes bind contain negatively charged phosphate backbones, it ispreferred that R₁₆ be substituted with one or more polar substituents.It is most preferred that R₁₆ include one or more positively chargedatoms in the polar substituent, such as is specified with regard to R₁₂above.

The cyanine dyes according to General Formula VI, i.e., dyes where apositively charged substituent is positioned off the nitrogen of thebenzothiazole portion of the dye, provide the advantage over previouscyanine dyes of exhibiting a significantly larger net fluorescenceenhancement with DNA than cyanine dyes where a positively chargedsubstituent is positioned at R₁₆ alone.

The use of intercalating dyes for staining cell nuclei requires that thedye itself be membrane-permeable or that a membrane permeabilizing stepbe incorporated into the sample preparation. In general, dyes with morethan one charge are not membrane permeable. Methods for enabling chargedmolecules and very large molecules into cells include the use ofchemicals, such as digitonin, freeze-thaw cell lysis steps, or the useof non-ionic detergents such as TRITON X-100. For speed and simplicity,it is preferred to add approximately 9 mM TRITON X-100.

The presence of a detergent solution (TRITON X-100) causes significantfluorescence enhancement of the dyes as compared to in PBS buffer. Anincrease in detergent-enhanced fluorescence (F_(TRITON) /F_(PBS)) hasthe effect of decreasing the net DNA enhanced fluorescence overdetergent-enhanced background fluorescence (F_(DNA) /F_(TRITON)). Thedetergent-enhanced fluorescence is believed to increase with increasinghydrophobicity.

                                      TABLE 3    __________________________________________________________________________    Fluorescence Ratios of Dyes in Buffer,    TRITON X-100 and DNA Solutions                                              F.sub.TRITON /F.sub.PBS                                                     F.sub.DNA /F.sub.PBS                                                           F.sub.DNA /F.sub.TR                                                           ITON    __________________________________________________________________________     ##STR31##                             6.sup.#                                              94     200   3     ##STR32##                             5* 12     100   8     ##STR33##                             7* 10     70    7     ##STR34##                             8  1.8    70    40    __________________________________________________________________________     *Compounds 5 and 7 are taught in U.S. Pat. No. 5,321,130 to Yue, et al.     .sup.# Compound 6 is taught in U.S. Pat. No. 4,957,870 to Lee, et al.

Fluorescence enhancement of the dyes upon binding to an excess of DNAwas found to be fairly constant regardless of how the quinolinium ringside chain was modified (R₁₆). Advantageously, however, it was foundthat inclusion of a positively charged substituent off the positivelycharged nitrogen of the benzothiazole portion of the dye (GeneralFormula VI) causes the dye to exhibit a significantly larger netDNA-enhancement than the positioning of a positively charged substituentat R₁₆ alone. As a result, smaller concentrations of nucleic acids canbe detected using cyanine dyes having General Formula VI.

For example, Table 3 compares the fluorescence ratios of dyes in asaline buffer, a detergent (TRITON X-100) and in a DNA solution. Dyesolutions (1.0 μM) were prepared in phosphate buffered saline (PBS), inPBS with TRITON X-100 (9 mM), and in PBS with double-stranded DNA (100μM).

Table 3 shows the effect of various side chains on the fluorescencebackground in TRITON X-100 (9 mM). As illustrated in Table 3, the netDNA enhanced fluorescence over detergent-enhanced backgroundfluorescence (F_(DNA) /F_(TRITON)) was found to be a factor of 5 greaterin dye 8 than in dye 7. This result is unexpected since the net chargeof 3+ is the same for both dyes 7 and 8. It appears that both thelocation and quantity of charges affect the fluorescence enhancement ofthe dyes.

The cyanine dyes according to General Formula VI preferably absorb lightat a wavelength of at least about 640 nm, more preferably at least about649 nm and emit fluorescence at a wavelength of at least about 650 nm,more preferably at least about 663 nm. The cyanine dyes also preferablyhave a positive Stoke's shift (λ_(Emission) -λ_(Abs).) of at least 11nm.

Table 4 provides examples of some of the preferred cyanine dyes havingGeneral Formula VI. It should be understood, however, that the dyeslisted in Table 4 are intended only to exemplify the cyanine dyes of thepresent invention and are not intended to be limiting.

    TABLE 4      -      ##STR35##      ##STR36##      ##STR37##      ##STR38##      ##STR39##      ##STR40##      ##STR41##      ##STR42##      ##STR43##      ##STR44##      ##STR45##      ##STR46##      ##STR47##      ##STR48##

The present invention also relates to the use of the cyanine dyes havingGeneral Formulas I, II, IV, V, VI, VII or VIII to form compositions fordetecting the presence of nucleic acids in a sample. In general, thecompositions include a cyanine dye according to the present inventionnon-covalently bound to a nucleic acid, i.e., DNA or RNA.

The fluorescence of the cyanine dyes of the present inventionsignificantly increase when bound to a nucleic acid. As a result, it ispossible to qualitatively or quantitatively determine the presence ofnucleic acids in a sample by monitoring the change in the fluorescenceintensity of the dye at a wavelength corresponding to the composition ofthe dye bound to the nucleic acids. Use of cyanine dyes in general fordetecting the presence of nucleic acids in a sample is known in the art.A discussion regarding the use of cyanine dyes to detect the presence ofnucleic acids in a sample is provided in U.S. Pat. No. 5,321,130 to Yue,et al. which is incorporated herein by reference.

The present invention also relates to a method for detecting nucleicacids by contacting the nucleic acids with a cyanine dye of the presentinvention. According to the method, a sample of nucleic acids arecontacted with a cyanine dye of the present invention in order to formthe composition of a cyanine dye non-covalently bound to a nucleic acidsequence. After the dye-nucleic acid sequence composition is formed, thebound dye is exposed to light having a wavelength near an absorbancemaximum of the dye when bound to a nucleic acid sequence. The resultingfluorescence emission of the dye is then detected in order toqualitatively or quantitatively determine the presence of nucleic acidsin the sample.

EXAMPLE 1

Preparation of Compound 4 ##STR49## 1a. Preparation of2,3-Tetramethylenenaphth[2,1-d]thiazolium Bromide ##STR50##

2-Aminonaphthalene-1-thiol was prepared by the method of Ambrogi, et al.(Ambrogi, V.; Grandolini, G.; Perioli, L.; Rossi, C. Synthesis, 992, 7,656-8.) 2-Aminonaphthalene-1-thiol (0.14 g, 0.8 mmol) and bromovalerylchloride (0.48 g, 2.4 mmol) were combined and heated to 100° for 1 h,then to 50° C. overnight. The resulting solid was washed with acetoneand air-dried to provide a white solid (0.16 g, 0.5 mmol, 60% yield).

1b. Preparation of IodoNAP6 ##STR51##

4-(2"-Acetanilidovinyl)-1'-(3'-iodopropyl)-quinolinium iodide (preparedby the general method of Brooker, et al. J. Am. Chem. Soc. 1941, 63,3192-3203; 32 mg, 63 μmol), 2,3-tetramethylenenaphth[2,1-d]thiazoliumbromide (20 mg, 63 μmol), triethylamine (40 μL) and ethanol (1 mL) werecombined and refluxed for 20 min. The dark blue solid was recrystallizedsequentially from isopropanol and ethanol to provide a purple solid (12mg, 30% yield). HPLC analysis on a C8 reverse-phase column usinggradient elution of 40% to 80% acetonitrile vs. 0.1M triethylammoniumacetate buffer showed one major peak at 16 min.

1c. Preparation of Compound 4 ##STR52##

IodoNAP6 (2 mg, 3 μmol) was dissolved in dimethylformamide.Trimethylamine was bubbled through the solution. The reaction wasmonitored by thin layer chromatography on silica gel with methanol asthe eluant. The Rf values of IodoNAP6 and compound 4 were 0.5 and zero,respectively. After 30 min, reaction was complete. The solvent wasevaporated and the residue partitioned between methylene chloride (CH₂Cl₂) and water. The aqueous layer was washed with 2×1 mL CH₂ Cl₂ andconcentrated to dryness. HPLC analysis with the same gradient that wasused with iodoNAP6 showed one broad peak at 7.2 min with no apparentstarting material. The absorbance maximum of compound 4 in methanol wasat 667 nm.

EXAMPLE 2

Preparation of Compound 8 ##STR53## 2a. Preparation of1',1"-(3',3"-Bisiodopropyl)-thia-4-carbocyanine Iodide ##STR54##

1'-(3'-Iodopropyl)-2-(2"-acetanilidovinyl)-benzothiazium iodide (15 mg,26 μmol), 1'-(3'-iodopropyl)-quinolinium iodide (15 mg, 34 μmol),triethylamine (50 μL) and methanol (1 mL) were combined at roomtemperature. A blue precipitate formed immediately. The reaction mixturewas centrifuged and the residue washed with methanol and isopropanol andair-dried to provide a dark solid (15 mg, 20 μmol, 77% yield).

2b. Preparation of Compound 8 ##STR55##

1',1"-(3',3'-Bisiodopropyl)-thia-4-carbocyanine iodide (15 mg, 20 μmol)was dissolved in DMF and trimethylamine bubbled through the solution.The reaction progress was monitored by TLC on reverse-phase plates with1:1 dimethylformamide:4M NaCl as eluant. The Rf's of the bisiodostarting material and the bisammonium salt were 0 and 0.8, respectively.The intermediate monoammonium salts could also be resolved, at Rf's of0.7 and 0.6. After 30 min the reaction was complete. The solvent wasevaporated. The absorbance maximum of compound 8 in DMSO was at 639 nm.

While the present invention is disclosed by reference to the preferredembodiments and examples detailed above, it is to be understood thatthese examples are intended in an illustrative rather than limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, which modifications will be within the spiritof the invention and the scope of the appended claims.

What is claimed is:
 1. A composition comprising: a cyclized fluorescentcyanine dye noncovalently bound to a nucleic acid polymer, the cyclizedcyanine dye having the general formula ##STR56## where: n is 0, 1 or 2;Yis selected from the group consisting of S or O; R₁ and R₂ are takentogether to form a 5, 6, 7 or 8 membered ring; R₃ and R₄ are eachindependently selected from the group consisting of hydrogen, C₁ -C₁₀alkyl, C₁ -C₁₀ alkoxy and C₁ -C₁₀ alkylthio; R₅ is a C₁ -C₅₀ alkyl; R₆and R₇ are each independently selected from the group consisting of H,and C₁₋₁₀ alkyl, or where R₆ and R₇ together to form a 5 or 6 memberedring; R₈ and R₉ are each independently selected from the groupconsisting of H and C₁₋₁₀ alkyl, or where R₈ and R₉ together to form a 5or 6 membered ring; and R₁₀ is selected from the group consisting of H,C₁₋₆ alkyl, and a fused benzene.
 2. The composition according to claim 1wherein dye includes a positively charged substituent attached to one ofthe atoms represented by R₁ and R₂ forming the 5, 6, 7 or 8 memberedring.
 3. The composition according to claim 2 wherein the positivelycharged substituent includes an aminoalkyl group.
 4. The compositionaccording to claim 3 wherein the aminoalkyl group includes a positivelycharged cyclic aminoalkyl group having a 1-5 positively charged nitrogenatoms.
 5. The composition according to claim 3 wherein the aminoalkylgroup has the general formula --R₂₈ N(R₂₉ R₃₀ R₃₁) where R₂₈ is a C₁₋₅alkyl and R₂₉, R₃₀ and R₃₁ are each independently a C₁₋₁₀ alkyl.
 6. Thecomposition of claim 1 wherein R₁ and R₂ are taken together to form a 7or 8 membered ring.
 7. The composition of claim 1 wherein R₅ issubstituted by at least one polar substituent.
 8. The composition ofclaim 7 wherein R₅ is substituted by at least one positively chargedatom.
 9. The composition of claim 8 wherein R₅ is an aminoalkyl chaincontaining a backbone of two to about 42 carbons and 1-5 positivelycharged nitrogens intermittently or equally spaced within the backbone,such that there are at least two carbons between sequential nitrogens.10. The composition of claim 9 wherein R₅ forms a 6-33 membered ring.11. The composition of claim 10 wherein R₅ is a positively chargedcyclic aminoalkyl group having 1-5 positively charged nitrogen atoms.12. The composition of claim 1 wherein n=1.
 13. The composition of claim1 wherein Y is S.
 14. The composition of claim 1 wherein R₆ and R₇ or R₈and R₉ are each H.
 15. The composition of claim 14 wherein R₆ and R₇ orR₈ and R₉ are taken together to form a 5 or 6 membered ring.
 16. Thecomposition of claim 15 wherein the ring formed by R₆ and R₇ or R₈ andR₉ is a 6 membered aromatic ring.
 17. The composition of claim 1 whereinR₁₀ is a fused benzene.
 18. A composition comprising: a cyclizedfluorescent cyanine dye noncovalently bound to a nucleic acid polymer,the cyclized cyanine dye having the general formula ##STR57## where: nis 0, 1 or 2;Y is selected from the group consisting of S or O; R₁ andR₂ are taken together to form a 5, 6, 7 or 8 membered ring; R₃ and R₄are each independently selected from the group consisting of hydrogen,C₁ -C₁₀ alkyl, C₁ -C₁₀ alkoxy and C₁ -C₁₀ alkylthio; R₅ is a C₁ -C₅₀alkyl; R₆ and R₇ are each independently selected from the groupconsisting of H, and C₁₋₁₀ alkyl, or where R₆ and R₇ together to form a5 or 6 membered ring; and R₁₀ is selected from the group consisting ofH, C₁₋₆ alkyl, C₁ -C₁₀ alkoxy and a fused benzene.
 19. The compositionaccording to claim 18 wherein dye includes a positively chargedsubstituent attached to one of the atoms represented by R₁ and R₂forming the 5, 6, 7 or 8 membered ring.
 20. The composition of claim 18wherein R₁ and R₂ are taken together to form a 7 or 8 membered ring. 21.The composition of claim 18 wherein R₅ is substituted by at least onepolar substituent.
 22. The composition of claim 21 wherein R₅ issubstituted by at least one positively charged atom.
 23. The compositionof claim 22 wherein R₅ is an aminoalkyl chain containing a backbone oftwo to about 42 carbons and 1-5 positively charged nitrogensintermittently or equally spaced within the backbone, such that thereare at least two carbons between sequential nitrogens.
 24. Thecomposition of claim 18 wherein n=1.
 25. The composition of claim 18wherein Y is S.
 26. The composition of claim 18 wherein R₆ and R₇ or R₈and R₉ are each H.
 27. The composition of claim 26 wherein R₆ and R₇ orR₈ and R₉ are taken together to form a 5 or 6 membered ring.
 28. Thecomposition of claim 27 wherein the ring formed by R₆ and R₇ or R₈ andR₉ is a 6 membered aromatic ring.
 29. The composition of claim 18wherein R₁₀ is a fused benzene.
 30. A method for detecting a nucleicacid polymer comprising:contacting a nucleic acid sequence with acyclized fluorescent cyanine dye to form a noncovalently bounddye-nucleic acid composition, the cyclized fluorescent cyanine dyehaving the general formula ##STR58## where n is 0, 1 or 2; Y is selectedfrom the group consisting of S or O; R₁ and R₂ are taken together toform a 5, 6, 7 or 8 membered ring; R₃ and R₄ are each independentlyselected from the group consisting of hydrogen, C₁ -C₁₀ alkyl, C₁ -C₁₀alkoxy and C₁ -C₁₀ alkylthio; R₅ is a C₁ -C₅₀ alkyl; R₆ and R₇ are eachindependently selected from the group consisting of H and C₁₋₁₀ alkyl,or where R₆ and R₇ together to form a 5 or 6 membered ring; R₈ and R₉are each independently selected from the group consisting of H and C₁₋₁₀alkyl, or where R₈ and R₉ together to form a 5 or 6 membered ring; andR₁₀ is selected from the group consisting of H, C₁₋₆ alkyl, C₁ -C₁₀alkoxy and a fused benzene; exposing the cyclized fluorescent cyaninedye bound to the nucleic acid polymer to light, the cyclized fluorescentcyanine dye absorbing the light and producing a fluorescence emission;and detecting the fluorescence emission.
 31. The method according toclaim 30 wherein dye includes a positively charged substituent attachedto one of the atoms represented by R₁ and R₂ forming the 5, 6, 7 or 8membered ring.
 32. The method of claim 30 wherein R₁ and R₂ are takentogether to form a 7 or 8 membered ring.
 33. The method of claim 30wherein n=1.
 34. The method of claim 30 wherein Y is S.
 35. The methodof claim 30 wherein R₆ and R₇ or R₈ and R₉ are each H.
 36. The method ofclaim 35 wherein R₆ and R₇ or R₈ and R₉ are taken together to form a 5or 6 membered ring.
 37. The method of claim 35 wherein the ring formedby R₆ and R₇ or R₈ and R₉ is a 6 membered aromatic ring.
 38. A methodfor detecting a nucleic acid polymer comprising:contacting a nucleicacid polymer with a cyclized fluorescent cyanine dye to form anoncovalently bound dye-nucleic acid polymer composition, the cyclizedfluorescent cyanine dye having the general formula ##STR59## where n is0, 1 or 2, Y is selected from the group consisting of S or O; R₁ and R₂are taken together to form a 5, 6, 7 or 8 membered ring, R₃ and R₄ areeach independently selected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₁ -C₁₀ alkoxy and C₁ -C₁₀ alkylthio, R₅ is a C₁ -C₅₀ alkyl,R₆ and R₇ are each independently selected from the group consisting of Hand C₁₋₁₀ alkyl, or where R₆ and R₇ together to form a 5 or 6 memberedring, R₈ and R₉ are each independently selected from the groupconsisting of H and C₁₋₁₀ alkyl, or where R₈ and R₉ together to form a 5or 6 membered ring and R₁₀ is selected from the group consisting of H,C₁₋₆ alkyl, C₁ -C₁₀ alkoxy and a fused benzene; exposing the cyclizedfluorescent cyanine dye bound to the nucleic acid polymer to light, thecyclized fluorescent cyanine dye absorbing the light and producing afluorescence emission; and detecting the fluorescence emission.